Methods and compositions relating to mesenchymal stem cell exosomes

ABSTRACT

The invention provides compositions comprising mesenchymal stem cell (MSC) derived exosomes, and methods of their use in subjects having certain lung diseases including inflammatory lung disease.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/867,816, filed Jan. 11, 2018, which is a continuation of U.S.application Ser. No. 14/004,237, filed Nov. 20, 2013, which is anational stage filing under 35 U.S.C. § 371 of International ApplicationNo. PCT/US2012/028524, filed Mar. 9, 2012, which claims priority to U.S.Provisional Patent Application filed Mar. 11, 2011, entitled “METHODSAND COMPOSITIONS RELATING TO MESENCHYMAL STEM CELL EXOSOMES”, Ser. No.61/451,981, the contents of which are incorporated by reference hereinin their entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers RO1

HL055454 and RO1 HL085446 awarded by the National Institutes of Health.The Government has certain rights in this invention.

BACKGROUND OF INVENTION

Premature infants suffer from or at risk of developing certain chroniclung (or respiratory) diseases (or conditions) at higher rates than fullterm or near term infants. Because the lungs and the breathing capacityof the infant are compromised, these diseases are often fatal. Theincreased survival rates of premature infants has led to an increasedincidence of such lung diseases. Inflammation is a keypathophysiological feature of multiple lung diseases including,pulmonary hypertension (PH or PAH), asthma, chronic obstructivepulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), andchronic lung disease of infancy, also known as bronchopulmonarydysplasia (BPD). The increased survival rates of premature infants hasled to an increased incidence of BPD and its associated complicationsthat include secondary PH, asthma, and increased rehospitalization ratein the first years of life. BPD is a common complication of prematurity(Kinsella et al., Lancet, 2006, 367:1421-1431; Stenmark and Abman, AnnuRev Physiol, 2005, 67:623-661) and in some studies, can affect up to35-40% of preterm infants born at <29 weeks gestation. Its underlyingcauses include mechanical injury, oxygen toxicity, infection, andresultant pulmonary inflammation and damage of the developing lung.Attempts to control BPD have involved gentle ventilation strategies anduse of anti-inflammatory agents such as corticosteroids. Thesetreatments however have limited success and unacceptable side effects(Baveja and Christou, Semin Perinatol, 2006, 30:209-218). Long-termeffects of these chronic lung diseases are also a concern and includesustained lung damage and neurodevelopmental delay. PH is a seriouscomplication of BPD and is associated with high mortality rate. It isalso associated with other forms of lung disease such as COPD. Morerecently, PH has been recognized to be a major complication ofschistosomiasis through mechanisms that involve inflammation.Schistosomiasis has very high prevalence in certain parts of the worldand is highly linked with secondary PH, potentially dramaticallyincreasing the incidence of this vascular disease worldwide.

SUMMARY OF INVENTION

The invention provides compositions comprising mesenchymal stem cell(MSC) derived exosomes and methods of use thereof in the treatmentand/or prevention of lung disease.

In one aspect, the invention provides a composition comprising isolatedmesenchymal stem cell (MSC) exosomes formulated for intratrachealadministration or administration by inhalation. In one aspect, theinvention provides a composition comprising isolated mesenchymal stemcell (MSC) exosomes formulated for intravenous administration.

In another aspect, the invention provides a composition comprisingisolated mesenchymal stem cell (MSC) exosomes and a pulmonarysurfactant.

In another aspect, the invention provides a composition comprisingisolated mesenchymal stem cell (MSC) exosomes and a pulmonarycorticosteroid. The pulmonary corticosteroid may be methylprednisolone,although it is not so limited.

In other aspects, the invention provides aerosolized isolatedmesenchymal stem cell (MSC) exosomes and compositions comprisingaerosolized isolated MSC exosomes.

In another aspect, the invention provides a composition of isolatedmesenchymal stem cell (MSC) exosomes for use in the treatment orprevention of lung disease. In another aspect, the invention provides apharmaceutical composition for use in the treatment or prevention oflung disease comprising isolated mesenchymal stem (MSC) exosomes.

In another aspect, the invention provides a composition of isolatedmesenchymal stem cell (MSC) exosomes for use as a medicament to treat orprevent lung disease.

In another aspect, the invention provides a method comprisingadministering to a subject having or at risk of developing a lungdisease an effective amount of isolated mesenchymal stem cell (MSC)exosomes.

In still another aspect, the invention provides use of isolatedmesenchymal stem cell (MSC) exosomes to treat or prevent lung disease ina subject, or use of isolated mesenchymal stem cell (MSC) exosomes inthe manufacture of a medicament for treating or preventing lung disease

In still another aspect, the invention provides isolated mesenchymalstem cell (MSC) exosomes for use in a method for treating or preventinglung disease comprising administering an effective amount of theisolated MSC exosomes to a subject having or at risk of developing lungdisease.

In another aspect, the invention provides a method comprisingadministering to a subject having or at risk of developing a lungdisease an effective amount of isolated mesenchymal stem cell (MSC)exosomes.

Various embodiments apply equally to the various aspects of theinvention, as described below. In some embodiments, the lung disease isinflammatory lung disease. In some embodiments, the inflammatory lungdisease is pulmonary hypertension, asthma, bronchopulmonary dysplasia(BPD), allergy, or idiopathic pulmonary fibrosis. In some embodiments,the lung disease is lung vascular disease. In some embodiments, the lungdisease is acute lung injury. In some embodiments, the acute lung injuryis associated with sepsis or is ventilator-induced acute respiratorydistress syndrome (ARDS).

In some embodiments, the subject has or is likely to developschistosomiasis.

In some embodiments, the subject is an neonate. In some embodiments, thesubject is an infant. In some embodiments, the subject is between 3-18years of age. In some embodiments, the subject is an adult. In any ofthese embodiments, the subject may be one that was born prematurely. Insome embodiments, the subject was born at less than 35 weeks ofgestation. In some embodiments, the subject was born at less than 26weeks of gestation.

In some embodiments, the isolated MSC exosomes are used together with asecondary agent. In some embodiments, the secondary agent is a steroid,an antioxidant, or inhaled nitric oxide. In some embodiments, thesteroid is a corticosteroid. In some embodiments, the corticosteroid ismethylprednisolone. In some embodiments, the antioxidant is superoxidedismutase.

In some embodiments, the isolated MSC exosomes are administered withinan hour of birth. In some embodiments, the isolated MSC exosomes areadministered within 1 month of birth.

In some embodiments, the isolated MSC exosomes are administeredintravenously. In some embodiments, the isolated MSC exosomes areadministered to lungs or trachea of the subject. In some embodiments,the isolated MSC exosomes are administered by inhalation. In someembodiments, the isolated MSC exosomes are administered in an aerosol.In some embodiments, the isolated MSC exosomes are administered using anebulizer. In some embodiments, the isolated MSC exosomes areadministered using an intratracheal tube.

In some embodiments, the isolated MSC exosomes are administered orformulated with a pulmonary surfactant. In some embodiments, thepulmonary surfactant is isolated naturally occurring surfactant. In someembodiments, the pulmonary surfactant is derived from bovine lung orporcine lung. In some embodiments, the pulmonary surfactant is asynthetic surfactant.

In some embodiments, the isolated MSC exosomes are administeredrepeatedly to the subject. In some embodiments, the isolated MSCexosomes are administered twice to the subject. In some embodiments, theisolated MSC exosomes are administered continuously to the subject.

In some embodiments, the isolated MSC exosomes are derived from cordblood MSC. In some embodiments, the isolated MSC exosomes are derivedfrom bone marrow MSC.

In some embodiments, the isolated MSC exosomes are autologous to thesubject. In some embodiments, the isolated MSC exosomes are allogeneicto the subject.

In some embodiments, the subject is not receiving a cell or organtransplantation.

Thus, in another aspect, the invention provides a pharmaceuticalcomposition comprising an effective amount of isolated human mesenchymalstem cell (MSC) exosomes and a pulmonary surfactant, formulated fordelivery to lungs, for use in a human subject having or at risk ofdeveloping a lung disease, wherein the subject is less than 4 weeks ofage. The invention similarly provides a method of use of the MSCexosomes comprising administering an effective amount of isolated humanmesenchymal stem cell (MSC) exosomes and a pulmonary surfactant,formulated for delivery to lungs, to a human subject having or at riskof developing a lung disease, wherein the subject is less than 4 weeksof age. The invention similarly provides use of an effective amount ofisolated human mesenchymal stem cell (MSC) exosomes and a pulmonarysurfactant, formulated for delivery to lungs, in a human subject havingor at risk of developing a lung disease, wherein the subject is lessthan 4 weeks of age.

In some embodiments, the isolated human MSC exosomes are isolated fromhuman umbilical cord (e.g., Wharton's Jelly). In some embodiments, thehuman subject was born before 37 weeks of gestation. In someembodiments, the human subject has been administered oxygen or has beenon a ventilator. In some embodiments, the human subject has or is atrisk of developing bronchopulmonary dysplasia. In some embodiments, thebronchopulmonary dysplasia is non-inflammatory. In some embodiments, theisolated human MSC exosomes are administered within 1 day of birth. Insome embodiments, the isolated human MSC exosomes are administeredwithin 1 hour of birth.

In another aspect, the invention provides synthetic MSC exosomes havingsimilar or identical characteristics of isolated MSC exosomes,compositions comprising such synthetic MSC exosomes, and methods oftheir use. The invention contemplates that synthetic MSC exosomes may beformulated and used in the same manner as isolated MSC exosomes. Thesynthetic exosomes may comprise one, two, three, four, five, six, sevenor all eight of the following proteins: haptoglobin (Acc. No. q61646),galectin-3-binding protein (Acc. No. q07797), thrombospondin-2 (Acc. No.q03350), lactadherin (Acc. No. q21956), adipocyte enhancer-bindingprotein 1 (Acc. No. q640n1), vimentin (Acc. No. p20152), proteasomesubunit alpha type 2 (Acc. No. p49′722), and amyloid beta A4 protein(Acc. No. p12023). These exosomes may be formulated as described hereinfor isolated MSC exosomes, including formulated for intranasal orintratracheal administration, or inhalation. They may be formulatedand/or administered with pulmonary surfactants or other therapeuticagents.

These and other aspects and embodiments of the invention will bedescribed in greater detail herein.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B. Secretory factors from BM-MSC are anti-inflammatory.Effects of BM-MSC-CM on hypoxia-induced infiltration of macrophages inthe lung (FIG. 1A). Mice (n>8) injected with either vehicle or BM-MSC-CMor MLF-CM were exposed to hypoxia (8.5% O₂) for 48 hours and BALFs fromthe hypoxic mice as well as age-matched normoxic control mice werecollected. Number of alveolar macrophages in BALFs were counted byKimura staining. Comparative immunoblot analysis of proteins incell-free BALFs from hypoxic and normoxic control mice. (FIG. 1B).Equivalent volume of cell-free BALFs from individual mouse in the samegroup (n>8) were pooled and proteins from equivalent volume of pooledBALFs were analyzed by western blot using antibodies specific for MCP-1(top) and HIMF/FIZZ1 (bottom). Relative intensities of MCP-1 and HIMFare represented by normalization over the IgA signal from the same blot.

FIGS. 2A-2F. Isolation of exosomes from cell-free BM-MSC-conditionedmedium. Exosomes in BM-MSC-CM or MLF-CM were isolated by ultrafiltrationand size-exclusion chromatography. 1.6% (w/w) of proteins in BM-MSC-CMwere associated with exosomes and exosomes in the fractions fromsize-exclusion chromatography were visualized by electron microscopy(FIGS. 2A-2D). To verify the isolation of exosomes from BM-MSC-CM, thefraction at void volume (v_(e)=v_(o)) (FIG. 2A) and the fraction betweenvoid volume and total volume (v_(o), <v_(e)<v_(t)) of the column (FIG.2B) were analyzed by negative staining electron microscopy at 30,000magnification. Morphology and size distribution of exosomes isolatedfrom BM-MSC-CM (FIG. 2C) or MLF-CM (FIG. 2D) were identical. Westernblot analysis against proteins associated with exosomes (FIGS. 2E-2F). 3μg proteins in each sample were assayed in western blot using theantibodies against CD63, 14-3-3s, moesin, macrophage colony stimulatingfactor (mCSF), osteopontin (OPN), and dicer. For positive control, 35 ugproteins of BM-MSC whole cell lysates were used.

FIGS. 3A-3B. MEX suppress hypoxia-induced acute pulmonary inflammation.Mice (n>7) injected with either vehicle or MEX or exosome-free fractionof BM-MSC-CM or FEX were exposed to hypoxia (8.5% 02) for 48 hours andBALFs of the hypoxic mice and age-matched normoxic mice were collected.Number of alveolar macrophages in BALF from each mouse was counted byKimura staining (FIG. 3A). Comparative immunoblot analysis of proteinsin cell-free BALFs from hypoxic and the normoxic control mice (FIG. 3B).Equivalent volume of cell-free BALFs from individual mouse in the samegroup (n>7) were pooled and proteins from equivalent volume of pooledBALFs were analyzed by western blot using antibodies specific for MCP-1(top) and HIMF/FIZZ1 (bottom). Relative levels of MCP-1 and HIMF arerepresented by normalization over the IgA signal from the same blot.

FIGS. 4A-4D. Time course effect on hypoxia-derived pulmonaryinflammation by single and multiple administration of MEX. Mice injectedwith either vehicle (FIG. 4A) or MEX (FIG. 4B) at day 0 were exposed tohypoxia (8.5% O₂) for the indicated periods. For multiple injectionexperiment, mice received MEX at day 0 were exposed to hypoxia for 4days. Second injection of the same dose of MEX at day 4 were followed byadditional exposure to hypoxia for the days indicated (FIG. 4C). BALFswere collected at selected time periods of hypoxic conditioning and thenumber of alveolar macrophages in BALF from individual mouse werecounted. Equivalent volume from cell-free BALFs of individual mouse inthe same group (n>7) were pooled and proteins from 10% (v/v) of pooledBALFs were analyzed by western blot using antibodies specific for MCP-1and HIMF (FIG. 4D).

FIGS. 5A-5E. MEX suppress hypoxia-induced PAH. Mice (n>7) were injectedonce or twice with either vehicle (at day 0 and day 4) or MEX (at day 0and/or day 4), or FEX (at day 0 and day 4) were exposed to hypoxia (8.5%O₂) for 3 week periods (FIG. 5A). RVSP (FIG. 5B) and Fulton's Index(FIG. 5C) of the hypoxic and normoxic control mice were measured at theend of experimental period. Paraffin embedded lung sections fromrandomly selected mice (n=4) in each group were immunostained for α-SMAto highlight pulmonary arterioles vessel walls (FIG. 5D). Originalmagnification for images: 400×. Small pulmonary arterioles with 20˜30 μmin diameter from each group were selected to measure vessel wallthickness which was expressed as a percentage of total vessel area. Dataare expressed as mean±SEM (n=40˜50 arterioles per group) (FIG. 5E).

FIGS. 6A-6D. Purification of MSCs-derived exosomes. Exosomes werepurified by Sephacryl S-400 gel filtration column chromatography.Negatively charged fluorescent 50 nm nanoparticles applied on the S-400column and eluted with identical condition to the exosome purification(FIG. 6A). From exosome purification, equivalent volume of each fractionwas separated on both 10% denaturing polyacrylamide gel (FIG. 6B) and1.2% agarose gel electrophoresis (FIG. 6C). Blot for the agarose gel wasstained with anti-CD81 antibody (FIG. 6D).

FIGS. 7A-7D. Comparative biochemical analysis of MSC-derived exosomesand exosome-free fractions. Equivalent protein quantities in both poolsof exosome fractions (M) and exosome-free fractions (MF) were separatedon denaturing 12% polyacrylamide gel (FIG. 7A). 1.2% agarose gel loadedby equivalent protein quantities in both pools of exosome fractions andexosome-free fractions as well as 50 nm nanoparticles in the absence(left, FIG. 7B) or presence of 0.5% SDS (right, FIG. 7B) were stainedwith colloidal blue. 1.2% agarose gel loaded by equivalent proteinquantities in both pools of exosome fractions and exosome-free fractionswere stained with ethidium bromide for nucleic acids (left, FIG. 7C) orcolloidal blue for proteins (right, FIG. 7C). Blots for gels loaded withequivalent protein quantities in both pools of exosome fractions andexosome-free fractions separated on both 1.2% agarose gel and 12%denaturing polyacrylamide gel were immunostained with anti-CD81 andanti-SPP-1 antibodies (FIG. 7D). M, Pool of exosomal fractions; MF, Poolof exosome-free fractions; N, negatively charged fluorescent 50 nmnanospheres.

FIGS. 8A-8B. Hypoxia-induced secretion of HIMF/FIZZ-1/Retnla in thelung. Mice were exposed over indicated time periods to monobaric hypoxia(8.5% 02). Proteins in BAL normalized by volume (FIG. 8A) and quantity(FIG. 8B) from each individual mouse in the same group were pooled andseparated on 14% polyacrylamide gel. Levels of HIMF, lysozyme, and IgAwere evaluated by western blot analysis using specific antibodies.

FIGS. 9A-9B. MSCs-derived exosomes suppress hypoxia-induced secretion ofHIMF/FIZZ-1/Retn1α in the lung. Mice injected either with 10 μg of MEX(M) or vehicle (V) by tail vein were exposed over indicated time periodsto monobaric hypoxia (8.5% 02). Proteins in BAL normalized by volume(FIG. 9A) and quantity (FIG. 9B) from each individual mouse in the samegroup were pooled and separated on 14% polyacrylamide gelelectrophoresis. Levels of HIMF, lysozyme, and IgA were evaluated bywestern blot analysis using specific antibodies.

FIGS. 10A-10C. MSCs-derived exosome suppress hypoxia-induced secretionof HIMF/FIZZ-1/Retn1α in the lung. Mice injected either with 10 μg ofMEX (M) or vehicle (V) by tail vein were exposed over indicated timeperiods to monobaric hypoxia (8.5% O₂). 10 μg BAL proteins from eachindividual mouse were separated on 14% polyacrylamide gelelectrophoresis (FIGS. 10A, 10B). Proteins in BAL normalized by quantityfrom each individual mouse in the same group were pooled and separatedon 14% polyacrylamide gel electrophoresis (FIG. 10C). Levels of HIMF,lysozyme, and IgA were evaluated by western blot analysis using specificantibodies.

FIGS. 11A-11C. MSCs-derived exosomes suppress hypoxia-inducedupregulation of HIF2α in the lung tissue. Mice injected with either 10μg MEX or vehicle by tail vein were exposed over indicated time periodsto monobaric hypoxia (8.5% O₂). Equivalent amount of proteins fromindividual lung tissue homogenate were separated on denaturingpolyacrylamide gel electrophoresis. Levels of HIF2α and actin weredetected by western blot analysis using specific antibodies (FIGS. 11A,11B). Relative intensities for HIF2α/actin were evaluated bydensitometric analysis (FIG. 11C). **, p<0.01 vs. normoxia (n=4±SD,One-way ANOVA); ##, p<0.01 vs. vehicle (hypoxia, 2 days) (n=4±SD,One-way ANOVA).

FIGS. 12A-12C. MSCs-derived exosomes suppress hypoxia-induced activationof NFkB p65 in the lung tissue. Mice injected with either 10 μg MEX orvehicle by tail vein were exposed over indicated time periods tomonobaric hypoxia (8.5% O₂). Equivalent amount of proteins fromindividual lung tissue homogenate were separated on denaturingpolyacrylamide gel electrophoresis. Levels of p65, phosporylated-p65(S536), and actin were detected by western blot analysis using specificantibodies (FIGS. 12A, 12B). Relative intensities for P-p65/actin wereevaluated by densitometric analysis (FIG. 12C). *, p<0.05 vs. normoxia(n=4±SD, One-way ANOVA); ##, p<0.01 vs. vehicle (hypoxia, 2 days)(n=4±SD, One-way ANOVA).

FIGS. 13A-13B. MSCs-derived exosomes suppress hypoxia-induced activationof STAT3 in the lung tissue. Mice injected with either 10 μg MEX orvehicle by tail vein were exposed to monobaric hypoxia (8.5% O₂) for 2days. Equivalent amount of proteins from individual lung tissuehomogenates were separated on denaturing polyacrylamide gelelectrophoresis. Levels of STAT3, phosporylated-STAT3 (Y705) and actinwere detected by western blot analysis using specific antibodies (FIG.13A). Relative intensities for P-STAT3/STAT3 were evaluated bydensitometric analysis (FIG. 13B). **, p<0.01 vs. normoxia (n=4±SD,One-way ANOVA); ##, p<0.01 vs. vehicle (n=4±SD, One-way ANOVA).

FIGS. 14A-14C. MSCs-derived exosomes suppress hypoxia-induced activationof STAT3 in the lung tissue. Mice injected with either 10 μg MEX orvehicle by tail vein were exposed over indicated time periods tomonobaric hypoxia (8.5% O₂). Equivalent amount of proteins fromindividual lung tissue homogenate were separated on denaturingpolyacrylamide gel electrophoresis. Levels of phosporylated-STAT3 (Y705)and actin were detected by western blot analysis using specificantibodies (FIGS. 14A, 14B). Relative intensities for P-STAT3/actin wereevaluated by densitometric analysis (FIG. 14C). ***, p<0.001 vs.normoxia or vehicle (hypoxia, 7 days), or MEX (hypoxia, 2 and 7 days)(n=4±SD, One-way ANOVA); ###, p<0.001 vs. vehicle (hypoxia, 2 days)(n=4±SD, One-way ANOVA); ns normoxia vs. MEX (hypoxia, 2 days) (n=4±SD,One-way ANOVA).

FIGS. 15A-15B. MSCs-derived exosomes suppress hypoxia-induced HIMFupregulation in the lung tissue. Mice injected with either 10 μg MEX orvehicle by tail vein were exposed to monobaric hypoxia (8.5% O₂) for 7days. Equivalent amount of proteins from individual lung tissuehomogenate were separated on denaturing polyacrylamide gelelectrophoresis. Levels of HIMF and actin were detected by western blotanalysis using specific antibodies (FIG. 15A). Relative intensities forHIMF/actin were evaluated by densitometric analysis (FIG. 15B). **,p<0.01 vs. normoxia (n=4±SD, One-way ANOVA); #, p<0.05 vs. vehicle (n=4±SD, One-way ANOVA); statistically non-significant between MEX vs.normoxia (n=4 ±SD, One-way ANOVA).

FIGS. 16A-16D. MSCs-derived exosomes suppress hypoxia-induced HIMFupregulation in the lung tissue. Mice injected with either 10 μg MEX orvehicle by tail vein were exposed over indicated time periods tomonobaric hypoxia (8.5% O₂). Equivalent amount of proteins fromindividual lung tissue homogenate were separated on denaturingpolyacrylamide gel electrophoresis. Levels of HIMF and actin weredetected by western blot analysis using specific antibodies (FIGS. 16A,16B). Relative intensities for HIMF/actin were evaluated bydensitometric analysis (FIGS. 16C, 16D). ***, p<0.001 vs. normoxia(n=4±SD, One-way ANOVA); **, p<0.01 vs. normoxia (n=4±SD, One-wayANOVA); #, p<0.05 vs. vehicle (n=4±SD, One-way ANOVA).

FIGS. 17A-17B. MSCs-derived exosomes protect chronic hypoxia-inducedright heart hypertrophy. Mice injected with either 10 μg MEX (M) orvehicle (V) by tail vein at indicated time periods were exposed tomonobaric hypoxia (8.5% O₂) for 3 weeks (FIG. 17A). Hearts fromindividual mouse were processed then ratio of RV/(LV+S) were measured(FIG. 17B). ***, p<0.001 vs. normoxia (n=9, One-way ANOVA); ###, p<0.001vs. vehicle (n=11, One-way ANOVA); statistically non-significant betweenMEX and normoxia (One-way ANOVA).

FIG. 18. High resolution profile of MSC exosome purification by FPLC(Fast Protein Liquid Chromatography). Upper panel: Fast Protein LiquidChromatography of MSC exosome purification. Matrix: HiPrep SephracylS-400. Mobile Phase Phosphate Buffered Saline, 300 mM. Flow rate: 0.5ml/min. Concentrated conditioned media were applied to the column andthe eluted protein was monitored by A280. Isolated MSC exosomes (MEX)eluted at 65.5 ml. A molecular size standard of nanoparticles of 50 nmdiameter co-eluted with MSC exosomes. Lower panel: Fractions of theeluated were applied to a native polyacrylamide electrophoresis gel andsubsequently stained for total protein. The MEX fraction migrated ashigh MW forms, distinct from bulk protein in the conditioned media.

FIGS. 19A-19B. MEX of either mouse or human origin suppress the hypoxicactivation of STAT3. (FIG. 19A) Total protein extracts from lungs ofindividual animals treated with 10 μg MEX preparations. Right Panel:Hypoxia exposure for 2 days resulted in activation of STAT3 throughphosphorylation at Tyr-705 (pY-STAT3) in mouse lung, and this wasprevented by treatment with MEX of mouse origin. Right panel:Quantitation of STAT3 activation. For all groups, n=4, One-way ANOVA:**, p<0.01 vs. Normoxia. **, p<0.01 vs. PBS. (FIG. 19B) Primary culturesof human Pulmonary Artery Endothelial Cells (hPAECs) exposed to hypoxia(1% O₂, 5 hrs) exhibit robust activation of STAT3 that is efficientlysuppressed in the presence of MEX secreted by MSCs from human umbilicalcord stroma (hUC-MEX). The microvesicle-depleted fraction of mediaconditioned by hUC-MSCs (hUC-ExD-CM) has no effect on STAT3 activation.

FIGS. 20A-20C. MEX treatment suppresses the hypoxic induction of themiR-17 microRNA superfamily and increases levels of anti-proliferativemiR-204 in the lung. MicroRNA levels in total mouse lung from animalstreated with 10 μg MEX preparations. miR levels were assessed by qPCR at7 days of hypoxic exposure and are presented relative to the mean of thenormoxic group. (FIG. 20A) Select miRs representing the miR-17-92,miR-106b˜25 and miR-106a˜363 clusters. (FIG. 20B) Select miRs reportedto be involved in hypoxic signaling. (FIG. 20C) Upregulation of basallevels of the pulmonary arteriole-specific miR-204 upon MEX treatment.Dots represent expression levels in individual animals. NRX: Normoxia;HPX: Hypoxia. For all groups, n=4, One-way ANOVA: **, p<0.01; ¶, p<0.001vs. Normoxia. §, p<0.001 vs. PBS.

FIG. 21. Schema of one non-limiting hypothesis synthesizing the resultsof this study. Hypoxia shifts the Th1/Th2 balance of immunomodulators inthe lung, resulting in alternative activated alveolar macrophages(AA-AMϕ and, in the early phase, induces the expression of HIMF in thelung epithelium. HIMF mitogenic action on the vasculature requires Th2cytokines, such as IL-4. Consequences of the shift towards proliferationinclude the hypoxic activation of STAT3 signaling and the upregulationof the miR-17 family of microRNAs. Treatment with MEX interferes with anearly hypoxic signal in the lung, suppressing both inflammation and HIMFtranscriptional upregulation. It addition, MEX treatment may directlyupregulate miR-204 levels, thus breaking the STAT3-miR-204-STAT3feed-forward loop, and shifting the balance to an anti-proliferativestate.

FIG. 22. Markers specific for exosomes from human Wharton's Jelly (WJ)MSCs. Western blot analysis of the 50 nm fraction (E1) from thefollowing sources: UC: unconditioned MSC growth media. MPD UC:microparticle-depleted growth media. Exosomal markers in the growthmedia are removed by polyethylene glycol precipitation. hMEX: exosomesfrom WJ MSCs. hFEX: exosomes from human dermal fibroblasts. TetraspaninsCD9 and CD81 are enriched in the exosomal fractions.

FIG. 23. mMEX suppresses hypoxic upregulation of HIF1α andphosphorylation of STAT3 in mouse lung fibroblasts. Mouse lungfibroblasts were exposed to hypoxia in the presence or absence of mousebone marrow MSC—derived exosomes (mMEX), as indicated. Hypoxia-induciblefactor (HIF) stabilization and STAT3 activation by phosphorylation(P-STAT3) were determined by western blotting.

FIG. 24. hPAEC treated with exosomes from mouse bone-marrow derived MSCs(mMEX, 1 ug/ml) or exosomes from mouse lung fibroblasts (mFEX ,1 ug/ml)were exposed to 1% O₂ for 6 hrs. Hypoxic activation of STAT3 (P-STAT3),total STAT3 and HIF2a stabilization was determined by western blotting.NRX: normoxia. PBS: hypoxia control.

FIG. 25. Human PAECs treated with exosomes from Wharton's Jelly MSCs(hMEX , 1 ug/ml) or exosomes from human dermal fibroblasts (hFEX, 1ug/ml) were exposed to 1% O₂ for 6 hrs. s Stat=3 activation (P-STAT3)and total STAT3 were determined by western blotting. NRX: normoxia. PBS:hypoxia control.

DETAILED DESCRIPTION OF INVENTION

The invention is based, in part, on the surprising finding that exosomesderived from mesenchymal stem cells provide therapeutic effect tocertain lung diseases including but not limited to inflammatory lungdiseases.

The invention relates broadly to compositions of mesenchymal stem cell(MSC) derived exosomes, which are interchangeably referred to asmesenchymal stem cell exosomes or MSC exosomes, and methods of their usein the treatment and/or prevention of certain lung diseases includingbut not limited to inflammatory lung diseases.

Exosomes and Exosome Preparation

The exosomes of the invention are membrane (i.e., lipid bilayer)vesicles that are released from mesenchymal stem cells. They have adiameter ranging from about 30 nm to 100 nm. By electron microscopy,exosomes appear to have a cup-shaped morphology. They sediment at about100,000×g and have a buoyant density in sucrose of about 1.10 to about1.21 g/ml. Exosomes may be referred to as microvesicles or nanovesicles.

Exosomes may comprise a number of proteins and/or nucleic acidsincluding RNA species such as miRNA. Proteins that may be expressed inexosomes include Alix, TSG101, CD63, CD9, CD81, moesin, HSP70, Dicer,M-CSF, osteopontin, and one or more of the proteins listed in Table 1(including any combination of 2, 3, 4, 5, 6, 7, or 8 of those proteinsalong with any of the proteins listed above). In some embodiments, theexosomes, including the synthetic exosomes discussed below, comprisemiRNA, Dicer, M-CSF, osteopontin, and one or more of the proteins ofTable 1 (including all of the proteins of Table 1).

Some aspects of the invention refer to isolated exosomes. As usedherein, an isolated exosome is one which is physically separated fromits natural environment. An isolated exosome may be physicallyseparated, in whole or in part, from tissue or cells with which itnaturally exists, including mesenchymal stem cells. In some embodimentsof the invention, a composition of isolated exosomes may be free ofcells such as mesenchymal stem cells, or it may be free or substantiallyfree of conditioned media. In some embodiments, the isolated exosomesmay be provided at a higher concentration than exosomes present inunmanipulated conditioned media.

Exosomes may be isolated from conditioned media from mesenchymal stemcell culture. A method for harvest of exosomes from mesenchymal stemcells is provided in the Examples. Briefly, such method involves firstculturing mesenchymal stem cells under standard conditions until theyreach about 70% confluency, and then culturing the cells in a serum-freemedia for 24 hours, following which the conditioned media is collectedand subjected to differential centrifugation at 400×g for 10 minutes and12000×g for 10 minutes in order to remove cells and cellular debris. Theclarified conditioned media is then concentrated by ultrafiltrationusing a 100 kDa MWCO filter (Millipore), and then centrifuged again at12000×g for 10 minutes. Exosomes are then isolated using size exclusionchromatography by loading the concentrated conditioned media on aPBS-equilibrated Chroma S-200 column (Clontech), eluting with PBS, andcollecting fractions of 350-550 microliters. Fractions containingexosomes are identified and potentially pooled. Protein concentration ismeasured using a standard Bradford assay (Bio-Rad). Aliquots of theenriched exosome preparations can be stored at −80° C.

Exosomes can also be purified by ultracentrifugation of clarifiedconditioned media at 100,000×g. They can also be purified byultracentrifugation into a sucrose cushion. GMP methods for exosomepurification from dendritic cells have been described in J ImmunolMethods. 2002;270:211-226.

Exosomes can also be purified by differential filtration, through nylonmembrane filters of defined pore size. A first filtration though a largepore size will retain cellular fragments and debris. A subsequentfiltration through a smaller pore size will retain exosomes and purifythem from smaller size contaminants.

The invention also contemplates the use of synthetic exosomes havingsome or all the characteristics of the isolated MSC exosomes describedherein. These synthetic exosomes would be synthesized in vitro (ratherthan derived and isolated from MSC or MSC-CM). They may be syntheticliposomes having one or more, including 2, 3, 4, 5, 6, 7, 8 or more ofthe proteins listed in Table 1 or FIG. 22. They may or may not comprisenucleic acids that encode one or more, including 2, 3, 4, 5, 6, 7, 8 ormore of these proteins. Liposome synthesis is known in the art, andliposomes may be purchased from commercial sources. It is to beunderstood that the various compositions, formulations, methods and usesdescribed herein relating to exosomes derived and isolated from MSC orMSC-CM are also contemplated in the context of synthetic exosomes.

The invention contemplates immediate use of exosomes or alternativelyshort- and/or long-term storage of exosomes, for example, in acryopreserved state prior to use. Proteinase inhibitors are typicallyincluded in freezing media as they provide exosome integrity duringlong-term storage. Freezing at −20° C. is not preferable since it isassociated with increased loss of exosome activity. Quick freezing at−80° C. is more preferred as it preserves activity. (See for exampleKidney International (2006) 69, 1471-1476.) Additives to the freezingmedia may be used in order to enhance preservation of exosome biologicalactivity. Such additives will be similar to the ones used forcryopreservation of intact cells and may include, but are not limited toDMSO, glycerol and polyethylene glycol.

TABLE 1 Specific and abundant proteins associated with MEX vs. FEXSequence MS/MS MS/MS coverage spectra Identification AccNo. spectra (%)in FEX Haptoglobin q61646 108 15.9 2 Galectin-3-binding protein q0779739 43.7 0 Thrombospondin-2 q03350 38 23.5 0 Lactadherin p21956 32 28.5 2Adipocyte enhancer-binding q640n1 27 19.1 3 protein 1 Vimentin p20152 2635.0 0 Proteasome subunit alpha type-2 p49722 26 50.9 3 Amyloid beta A4protein p12023 25 27.1 2 * Data were presented when total MS/MS hitsare >25 and the ratio of MEX/FEX in sequence coverage is >3 for theparticular protein.

Mesenchymal Stem Cells

A mesenchymal stem cell is a progenitor cell having the capacity todifferentiate into neuronal cells, adipocytes, chondrocytes,osteoblasts, myocytes, cardiac tissue, and other endothelial andepithelial cells. (See for example Wang, Stem Cells 2004;22(7);1330-7;McElreavey;1991 Biochem Soc Trans (1);29s; Takechi, Placenta 1993March/April; 14 (2); 235-45; Takechi, 1993; Kobayashi; Early HumanDevelopment;1998; July 10; 51 (3); 223-33;

Yen; Stem Cells; 2005; 23 (1) 3-9.) These cells may be definedphenotypically by gene or protein expression. These cells have beencharacterized to express (and thus be positive for) one or more of CD13,CD29, CD44, CD49a, b, c, e, f, CD51, CD54, CD58, CD71, CD73, CD90,CD102, CD105, CD106, CDw119, CD120a, CD120b, CD123, CD124, CD126, CD127,CD140a, CD166, P75, TGF-bIR, TGF-bIIR, HLA-A, B, C, SSEA-3, SSEA-4, D7and PD-L1. These cells have also been characterized as not expressing(and thus being negative for) CD3, CD5, CD6, CD9, CD10, CD11a, CD14,CD15, CD18, CD21, CD25, CD31, CD34, CD36, CD38, CD45, CD49d, CD50,CD62E, L, S, CD80, CD86, CD95, CD117, CD133, SSEA-1, and ABO. Thus,mesenchymal stem cells may be characterized phenotypically and/orfunctionally according to their differentiative potential.

Mesenchymal stem cells may be harvested from a number of sourcesincluding but not limited to bone marrow, blood, periosteum, dermis,umbilical cord blood and/or matrix (e.g., Wharton's Jelly), andplacenta. Methods for harvest of mesenchymal stem cells are described ingreater detail in the Examples. Reference can also be made to U.S. Pat.No. 5,486,359 for other harvest methods that can be used in the presentinvention.

The mesenchymal stem cells, and thus the exosomes, contemplated for usein the methods of the invention may be derived from the same subject tobe treated (and therefore would be referred to as autologous to thesubject) or they may be derived from a different subject preferably ofthe same species (and therefore would be referred to as allogeneic tothe subject).

As used herein, it is to be understood that aspects and embodiments ofthe invention relate to cells as well as cell populations, unlessotherwise indicated. Thus, where a cell is recited, it is to beunderstood that a cell population is also contemplated unless otherwiseindicated.

As used herein, an isolated mesenchymal stem cell is a mesenchymal stemcell that has been physically separated from its natural environment,including physical separation from one or more components of its naturalenvironment. Thus, an isolated cell or cell population embraces a cellor a cell population that has been manipulated in vitro or ex vivo. Asan example, isolated mesenchymal stem cells may be mesenchymal stemcells that have been physically separated from at least 50%, preferablyat least 60%, more preferably at least 70%, and even more preferably aleast 80% of the cells in the tissue from which the mesenchymal stemcells are harvested. In some instances, the isolated mesenchymal stemcells are present in a population that is at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% mesenchymal stem cells as phenotypically and/or functionallydefined herein. Preferably the ratio of mesenchymal stem cells to othercells is increased in the isolated preparation as compared to thestarting population of cells.

Mesenchymal stem cells can be isolated using methods known in the art,e.g., from bone marrow mononuclear cells, umbilical cord blood, adiposetissue, placental tissue, based on their adherence to tissue cultureplastic. For example, mesenchymal stem cells can be isolated fromcommercially available bone marrow aspirates. Enrichment of mesenchymalstem cells within a population of cells can be achieved using methodsknown in the art including but not limited to FACS.

Commercially available media may be used for the growth, culture andmaintenance of mesenchymal stem cells. Such media include but are notlimited to Dulbecco's modified Eagle's medium (DMEM). Components in suchmedia that are useful for the growth, culture and maintenance ofmesenchymal stem cells include but are not limited to amino acids,vitamins, a carbon source (natural and non-natural), salts, sugars,plant derived hydrolysates, sodium pyruvate, surfactants, ammonia,lipids, hormones or growth factors, buffers, non-natural amino acids,sugar precursors, indicators, nucleosides and/or nucleotides, butyrateor organics, DMSO, animal derived products, gene inducers, non-naturalsugars, regulators of intracellular pH, betaine or osmoprotectant, traceelements, minerals, non-natural vitamins. Additional components that canbe used to supplement a commercially available tissue culture mediuminclude, for example, animal serum (e.g., fetal bovine serum (FBS),fetal calf serum (FCS), horse serum (HS)), antibiotics (e.g., includingbut not limited to, penicillin, streptomycin, neomycin sulfate,amphotericin B, blasticidin, chloramphenicol, amoxicillin, bacitracin,bleomycin, cephalosporin, chlortetracycline, zeocin, and puromycin), andglutamine (e.g., L-glutamine). Mesenchymal stem cell survival and growthalso depends on the maintenance of an appropriate aerobic environment,pH, and temperature. Mesenchymal stem cells can be maintained usingmethods known in the art. (See for example Pittenger et al., Science,284:143-147 (1999).)

Subjects

The methods of the invention may be performed on any subject likely toderive benefit therefrom, including human subjects, agriculturallivestock (e.g., cows, pigs, etc.), prized animals (e.g., horses),companion animals (e.g., dogs, cats, etc.), and the like. In variousaspects of the invention, human subjects are preferred. In some aspect,human subjects and human MSC exosomes are used.

The subjects may be those that have a lung disease (or condition)amenable to treatment using the exosomes of the invention, or they maybe those that are at risk of developing such a disease (or condition).Such subjects include neonates and particularly neonates born at lowgestational age. As used herein, a human neonate refers to an human fromthe time of birth to about 4 weeks of age. As used herein, a humaninfant refers to a human from about the age of 4 weeks of age to about 3years of age. As used herein, low gestational age refers to birth (ordelivery) that occurs before a normal gestational term for a givenspecies. In humans, a full gestational term is about 40 weeks and mayrange from 37 weeks to more than 40 weeks. Low gestational age, inhumans, akin to a premature birth is defined as birth that occurs before37 weeks of gestation. The invention therefore contemplates preventionand/or treatment of subjects born before 37 weeks of gestation,including those born at even shorter gestational terms (e.g., before 36,before 35, before 34, before 33, before 32, before 31, before 30, before29, before 28, before 27, before 26, or before 25 weeks of gestation).Typically such premature infants will be treated as neonates, howeverthe invention contemplates their treatment even beyond the neonate stageand into childhood and/or adulthood. Certain subjects may have a geneticpredisposition to certain forms of lung disease such as for examplepulmonary hypertension, and those subjects may also be treated accordingto the invention.

Methods of Preventing and Treating Diseases

The invention contemplates preventing and treating certain lungdiseases. Preventing a disease means reducing the likelihood that thedisease manifests itself and/or delaying the onset of the disease.Treating a disease means reducing or eliminating the symptoms of thedisease.

The invention intends to prevent and/or treat a number of lung (orpulmonary) diseases. These diseases include inflammatory lung diseasessuch as but not limited to pulmonary hypertension (PH) which is alsoreferred to as pulmonary artery hypertension (PAH), asthma,bronchopulmonary dysplasia (BPD), allergies, sarcoidosis, and idiopathicpulmonary fibrosis. These diseases also include lung vascular diseaseswhich may not have an inflammatory component. Still other pulmonaryconditions that may be treated according to the invention include acutelung injury which may be associated with sepsis or with ventilation. Anexample of this latter condition is acute respiratory distress syndromewhich occurs in older children and adults.

Pulmonary hypertension is a lung disease characterized by blood pressurein the pulmonary artery that is far above normal levels. Symptomsinclude shortness of breath, chest pain particularly during physicalactivity, weakness, fatigue, fainting, light headedness particularlyduring exercise, dizziness, abnormal heart sounds and murmurs,engorgement of the jugular vein, retention of fluid in the abdomen, legsand ankles, and bluish coloring in the nail bed.

Bronchopulmonary dysplasia is a condition that afflicts neonates whohave been given oxygen or have been on ventilators, or neonates bornprematurely particularly those born very prematurely (e.g., those bornbefore 32 weeks of gestation). It is also referred to as neonatalchronic lung disease. Causes of BPD include mechanical injury forexample as a result of ventilation, oxygen toxicity for example as aresult of oxygen therapy, and infection. The disease may progress fromnon-inflammatory to inflammatory with time. Symptoms include bluishskin, chronic cough, rapid breathing, and shortness of breath. Subjectshaving BPD are more susceptible to infections such as respiratorysyncytial virus infection. Subjects having BPD may develop pulmonaryhypertension.

Acute respiratory distress syndrome (ARDS), also known as respiratorydistress syndrome (RDS) or adult respiratory distress syndrome is acondition that arises as a result of injury to the lungs or acuteillness. The injury to the lung may be a result of ventilation, trauma,burns, and/or aspiration. The acute illness may be infectious pneumoniaor sepsis. It is considered a severe form of acute lung injury, and itis often fatal. It is characterized by lung inflammation, impaired gasexchange, and release of inflammatory mediators, hypoxemia, and multipleorgan failure. ARDS can also be defined as the ratio of arterial partialoxygen tension (PaO₂) as a fraction of inspired oxygen (FiO₂) below 200mmHg in the presence of bilateral infiltrates on the chest x-ray. APaO_(2/)FiO₂ ratio less than 300 mmHg with bilateral infiltratesindicates acute lung injury, which is often a precursor to ARDS.Symptoms of ARDS include shortness of breath, tachypnea, and mentalconfusion due to low oxygen levels.

Idiopathic pulmonary fibrosis is characterized by scarring or thickeningof the lungs without a known cause. It occurs most often in persons50-70 years of age. Its symptoms include shortness of breath, regularcough (typically a dry cough), chest pain, and decreased activity level.

Prevention and/or treatment may involve in some instances use of the MSCexosomes alone or together with one or more secondary agents. Subjectsmay also be subjected to mechanical interventions such as ventilationwith or without exogenous oxygen administration.

With respect to neonates and particularly low gestation age neonates,the invention contemplates administration of MSC exosomes within 4weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days,1 day, 12 hours, 6 hours, 3 hours, or 1 hour of birth. In some importantinstances, the MSC exosomes are administered within 1 hour of birth.

The invention further contemplates administration of MSC exosomes evenin the absence of symptoms indicative of a pulmonary disease such as butnot limited to BPD.

The invention also contemplates repeated administration of MSC exosomes,including two, three, four, five or more administrations of MSCexosomes. In some instances, the MSC exosomes may be administeredcontinuously. Repeated or continuous administration may occur over aperiod of several hours (e.g., 1-2, 1-3, 1-6, 1-12, 1-18, or 1-24hours), several days (e.g., 1-2, 1-3, 1-4, 1-5, 1-6 days, or 1-7 days)or several weeks (e.g., 1-2 weeks, 1-3 weeks, or 1-4 weeks) depending onthe severity of the condition being treated. If administration isrepeated but not continuous, the time in between administrations may behours (e.g., 4 hours, 6 hours, or 12 hours), days (e.g., 1 day, 2 days,3 days, 4 days, 5 days, or 6 days), or weeks (e.g., 1 week, 2 weeks, 3weeks, or 4 weeks). The time between administrations may be the same orthey may differ. As an example, if the symptoms of the disease appear tobe worsening the MSC exosomes may be administered more frequently, andthen once the symptoms are stabilized or diminishing the MSC exosomesmay be administered less frequently.

In some important instances, the MSC exosomes are administered at leastonce within 24 hours of birth and then at least once more within 1 weekof birth. Even more preferably, the MSC exosomes are administered atleast once within 1 hour of birth and then at least once more within 3-4days of birth.

In some instances, repeated intravenous administration of low doses ofMSC exosomes may occur. It has been found in accordance with theinvention that when low doses of MSC exosomes were administeredintravenously to murine subjects, maximal activity was achieved when theMSC exosomes were administered every 2-4 days. In these experiments, 100ng of MSC exosomes were administered to on average a 20 gram mouse,corresponding to a dose of 5 micrograms per kilogram. When higher doseswere used (e.g., 10 micrograms per 20 gram mouse or 0.5 milligrams perkilogram), a single intravenous administration was sufficient to achievelong-term protection. Accordingly, the invention contemplates repeatedadministration of low dosage forms of MSC exosomes as well as singleadministrations of high dosage forms of MSC exosomes. Low dosage formsmay range from, without limitation, 1-50 micrograms per kilogram, whilehigh dosage forms may range from, without limitation, 51-1000 microgramsper kilogram. It will be understood that, depending on the severity ofthe disease, the health of the subject, and the route of administration,inter alia, the single or repeated administration of low or high doseMSC exosomes are contemplated by the invention.

Administration, Pharmaceutical Compositions, Effective Amounts

The MSC exosomes may be used (e.g., administered) in pharmaceuticallyacceptable preparations (or pharmaceutically acceptable compositions),typically when combined with a pharmaceutically acceptable carrier. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, and may optionally comprise other (i.e., secondary)therapeutic agents.

A pharmaceutically acceptable carrier is a pharmaceutically acceptablematerial, composition or vehicle, such as a liquid or solid filler,diluent, excipient, solvent or encapsulating material, involved incarrying or transporting a prophylactically or therapeutically activeagent. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Some examples of materials which can serve aspharmaceutically acceptable carriers include sugars, such as lactose,glucose and sucrose; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; buffering agents, such as magnesiumhydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; phosphate buffer solutions; and othernon-toxic compatible substances employed in pharmaceutical formulations.

Secondary Therapeutic Agents. The exosomes may be administered with oneor more secondary therapeutic agents. As used herein, a therapeuticagent refers to any agent which can be used in the prevention, treatmentand/or management of a lung disease such as those discussed herein.These include but are not limited to surfactants, inhaled nitric oxide,almitrine bismesylate, immunomodulators, and antioxidants. Examples ofimmunomodulators include steroids and corticosteroids such as but notlimited to methylprednisolone. Examples of antioxidants include but arenot limited to superoxide dismutase.

Certain secondary therapeutic agents used in the treatment or managementof certain lung diseases including but not limited to pulmonaryhypertension include oxygen, anticoagulants such as warfarin (Coumadin);diuretics such as furosemide (Lasix®) or spironalactone)(Aldactone®);calcium channel blockers; potassium such as K-dur®; inotropic agentssuch as digoxin; vasodilators such as nifedipine (Procardia® ordiltiazem (Cardizem®); endothelin receptor antagonists such asbosentan)(Tracleer® and ambrisentan)(Letairis® ; prostacyclin analoguessuch as epoprostenol (Flolan®), treprostinil sodium (Remodulin®,Tyvase), and iloprost (Ventavie); and PDE-5 inhibitors such assildenafil (Revatio®) and tadalafil (Adcirca®).

Surfactants. The MSC exosomes may be administered with pulmonarysurfactants. A pulmonary surfactant is a lipoprotein mixture useful inkeeping lung airways open (e.g., by preventing adhesion of alveolarwalls to each other). Pulmonary surfactants may be comprised ofphospholipids such as dipalmitoylphosphatidylcholine (DPPC),phosphotidylcholine (PC), phosphotidylglycerol (PG); cholesterol; andproteins such as SP-A, B, C and D. Pulmonary surfactants may be derivedfrom naturally occurring sources such as bovine or porcine lung tissue.Examples include Alveofact™ (from cow lung lavage), Curosurf™ (fromminced pig lung), Infasurf™ (from calf lung lavage), and Survanta™ (fromminced cow lung, with additional components including DPPC, palmiticacid, and tripalmitin). Pulmonary surfactants may also be synthetic.Examples include Exosurf™ (comprised of DPPC with hexadecanol andtyloxapol), Pumactant™ or Artificial Lung Expanding Compound (ALEC)(comprised of DPPC and PG), KL-4 (comprised of DPPC, palmitoyl-oleoylphosphatidylglyercol, palmitic acid, and synthetic peptide that mimicsSP-B), Venticute™ (comprised of DPPC, PG, palmitic acid, and recombinantSP-C). Pulmonary surfactants may be obtained from commercial suppliers.

Effective Amounts. The preparations of the invention are administered ineffective amounts. An effective amount is that amount of an agent thatalone stimulates the desired outcome. The absolute amount will dependupon a variety of factors, including the material selected foradministration, whether the administration is in single or multipledoses, and individual patient parameters including age, physicalcondition, size, weight, and the stage of the disease. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation.

Administration Route. The MSC exosomes may be administered by any routethat effects delivery to the lungs. Systemic administration routes suchas intravenous bolus injection or continuous infusion are suitable. Moredirect routes such as intranasal administration, intratrachealadministration (e.g., via intubation), and inhalation (e.g., via anaerosol through the mouth or nose) are also contemplated by theinvention and in some instances may be more appropriate particularlywhere rapid action is necessary. As used herein, an aerosol is asuspension of liquid dispersed as small particles in a gas, and itincludes a fine mist or a spray containing such particles. As usedherein, aerosolization is the process of producing of an aerosol bytransforming a liquid suspension into small particles or droplets. Thismay be done using an aerosol delivery system such as a pressurized packor a nebulizer. Nebulizers include air-jet (i.e., pneumatic),ultrasonic, and vibrating-mesh nebulizers, for example with the use of asuitable propellant such as but not limited to dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In addition to nebulizers, other devices forpulmonary delivery include but are not limited to metered dose inhalers(MDIs) and dry powder inhalers (DPIs). Capsules and cartridges of forexample gelatin for use in an inhaler or insufflator may be formulatedcontaining lyophilized exosomes and a suitable powder base such aslactose or starch.

The exosomes, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, including forexample by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with or without an added preservative.

The compositions may take such forms as water-soluble suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Suitable lipophilic solvents or vehicles include fatty oils suchas sesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension, such as sodiumcarboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents whichincrease solubility. Alternatively, the exosomes may be in lyophilizedor other powder or solid form for constitution with a suitable vehicle,e.g., sterile pyrogen-free water, before use.

It is to be understood that other agents to be administered to subjectsbeing treated according to the invention may be administered by anysuitable route including oral administration, intranasal administration,intratracheal administration, inhalation, intravenous administration,etc. Those of ordinary skill in the art will know the customary routesof administration for such secondary agents.

Kits

The invention also encompasses a packaged and labelled pharmaceuticalproduct. This article of manufacture or kit includes the appropriateunit dosage form in an appropriate vessel or container such as a glassvial or plastic ampoule or other container that is hermetically sealed.The unit dosage form should be suitable for pulmonary delivery forexample by aerosol. Preferably, the article of manufacture or kitfurther comprises instructions on how to use including how to administerthe pharmaceutical product. The instructions may further containinformational material that advises a medical practitioner, technicianor subject on how to appropriately prevent or treat the disease ordisorder in question. In other words, the article of manufactureincludes instructions indicating or suggesting a dosing regimen for useincluding but not limited to actual doses, monitoring procedures, andother monitoring information.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment.

The kits may include MSC exosomes in sterile aqueous suspensions thatmay be used directly or may be diluted with normal saline forintravenous injection or use in a nebulizer, or dilution or combinationwith surfactant for intratracheal administration. The kits may thereforealso contain the diluent solution or agent, such as saline orsurfactant. The kit may also include a pulmonary delivery device such asa nebulizer or disposable components therefore such as the mouthpiece,nosepiece, or mask.

EXAMPLES Summary

Hypoxia induces an inflammatory response in the lung manifested byalternative activation of macrophages with elevation of pro-inflammatorymediators that are critical for the later development of hypoxicpulmonary hypertension (HPH). Mesenchymal stromal cell (MSC)transplantation prevents lung inflammation, vascular remodeling andright heart failure, and inhibits HPH in experimental models of disease.In this study, we aimed to investigate the paracrine mechanisms by whichMSCs are protective in HPH.

We fractionated mouse MSC-conditioned media to identify thebiologically-active component affecting in vivo hypoxic signaling anddetermined that exosomes, secreted membrane microvesicles, suppressedthe hypoxic pulmonary influx of macrophages and the induction ofpro-inflammatory and pro-proliferative mediators, including monocytechemoattractant protein-1 and hypoxia-inducible mitogenic factor, in themurine model of HPH. Intravenous delivery of MSC exosomes (MEX)prevented vascular remodeling and development of HPH. Multipleadministrations of low dose MEX completely suppressed early hypoxiainflammatory response and ameliorated pulmonary hypertension and rightventricular pathology. A single high dose of MEX was found to besufficient for preventing vascular remodeling and development of PHinduced by chronic hypoxia. In contrast, fibroblast-derived exosomes andMEX-depleted media had no effect. MEX suppressed the hypoxic activationof signal transducers and activators of transcription 3 (STAT3) and theupregulation of the miR-17 superfamily of microRNA clusters, whereas itincreased lung levels of miR-204, a key microRNA whose expression isdecreased in human PH. MEX produced by human umbilical cord MSCsinhibited STAT3 signaling in isolated human PAECs, demonstrating adirect effect of MEX on hypoxic STAT3 activation.

This study indicates that MEX exert a pleiotropic protective effect onthe lung and can prevent PH through suppression of specificSTAT3-mediated hyperproliferative pathways induced by hypoxia.

Materials and Methods

Isolation of bone marrow-derived mesenchymal stem cells. Bonemarrow-derived mesenchymal stem cells (BM-MSCs) were isolated from thefemurs and tibiae of 5-7 week old FVB/s mice as previously described.Briefly, the ends of each tibia and femur were clipped to expose themarrow and the bones inserted into adapted centrifuge tubes. The tubeswere centrifuged for 1 minute at 400×g to collect the marrow. The pelletwas resuspended in 3 mL α-minimal essential medium (α-MEM) mediumthrough a 21-gauge needle followed by filtration through a 70-μm nylonmesh filter. The marrow cells were layered on a Ficoll-Paque (Amersham)density gradient, centrifuged and plated. Plastic adherent cells weremaintained in culture with media changed every 2-3 days. Following 2-3passages, immunodepletion was performed as per published protocols andthe International Society for Cellular Therapy (ISCT) guidelines¹. Thecells were negatively selected for CD11b, CD14, CD19, CD31, CD34, CD45,and CD79α antigens using the appropriate fluorescent-tagged antibodies(BD Biosciences) in a fluorescence-activated cell sorter (MoFlo),further propagated, and then positively selected for CD73, CD90, CD105,c-kit and Sca-1 antigens, as above. All reagents were purchased fromSigma. Isolated cells between passages 7-12 can be used for theproduction of conditioned medium and for the isolation of exosomes.Isolated and/or cultured cells may also be cryopreserved prior toproduction of conditioned medium or exosomes.

Isolation of primary mouse lung fibroblast. Primary mouse lungfibroblast (MLF) cultures were derived according to standard methods.

Preparation of MSC-conditioned medium (MSC-CM). Cryo-preserved MSCs wereplated with complete medium (αMEM (Invitrogen) supplemented with 10% FBS(Hyclone), 10% Horse serum (Hyclone), and 5 mM L-glutamine (Gibco))followed by incubation under standard culture conditions. Serum-freeMSC-CM produced for 24 hrs from the culture was clarified bydifferential centrifugation at 400×g for 10 min and 12,000×g for 20 min.Serum-free MSC-CM was concentrated 250 times by ultrafiltration with 100kDa MWCO filter devices (Millipore) followed by further clarification bycentrifugation at 12,000×g for 20 min.

Purification of exosomes by Sephacryl 5-400 gel filtrationchromatography. 250× concentrates of MSC-CM was applied on S-400 column(14×300 mm, Pharmacia) pre-equilibrated with PB2XS buffer (20 mM sodiumphosphate buffer (pH 7.4) supplemented with 300 mM NaC1) and eluted withconstant flow rate (0.4 ml/min). Equivalent volume from each fraction(0.8 ml) was applied on denaturing 10% polyacrylamide gel or native 1.2%agarose gel followed by immuno-staining with specific antibodies againstCD81 (Santa Cruz) and SPP-1 (Osteopontin) (R&D Systems). Fractionspositive for both CD81 and SPP-1 with higher migration in native agarosegel were pooled and used as an exosome preparation (FIG. 1). Pooledexosomes could be used immediately or snap frozen in liquid nitrogen andthen stored at −80° C.

Electron microscopic analysis. Purified exosomes were adsorbed to acarbon coated grid that had been made hydrophilic by a 30 secondexposure to a glow discharge. Excess liquid was removed and the exosomeswere stained with 0.75% uranyl formate for 30 seconds. After removingthe excess uranyl formate, the grids were examined in a JEOL 1200EXTransmission electron microscope and images were recorded with an AMT 2kCCD camera.

Proteomic analysis of exosomes. 30 μg of exosomal proteins wereseparated on 12% denaturing PAGE and subsequently digested withsequencing grade trypsin (Promega). The sequence analysis was performedat the Harvard Microchemistry and Proteomics Analysis Facility bymicrocapillary reverse-phase HPLC nano-electrospray tandem massspectrometry (pLC/MS/MS) on a Thermo LTQ-Orbitrap mass spectrometer. Theresulting MS/MS spectra of the peptides were then correlated withspecies specific sequences using the algorithm SEQUEST and programsdeveloped in the Harvard Microchemistry Facility.

Western blot analysis. In experiments for characterizing exosomes, 3 μgproteins from either exosomal fractions or exosome free fraction wereseparated on 12% polyacrylamide gel electrophoresis following transferto 0.45 μm PVDF membrane (Millipore). After blocking with 5% skim milk,specific signals were detected using polyclonal goat anti-CD63 (SantaCruz), anti-CD81 (Santa Cruz), anti-mCSF (R & D systems),anti-osteopontin (R & D systems), polyclonal rabbit anti-moesin (Abcam),anti-14-3-3 family (Abcam), and monoclonal anti-Dicer (Abcam) withappropriate peroxidase-conjugated secondary antibodies. For the control,35 μg proteins of BM-MSC extract were used in parallel. For analysis ofproteins in BALFs, Equivalent volume of cell-free BALF from individualmouse in the same group were pooled then following precipitationovernight by 20% trichloroacetic acid (TCA). The protein pelletsresuspended in lx sodium lauryl sulfate (SDS)-loading buffer were thenseparated on denaturing tris-tricine polyacrylamide gel. After transferto 0.2 μm PVDF membranes (Millipore), blots were blocked with 5% skimmilk in PBS containing 0.1% tween 20 (Sigma) for 1 hour followingincubation with 1:1,000 diluted rabbit polyclonal anti-monocytechemoattractant protein-1 (MCP-1) antibody (Abcam), anti-hypoxia-inducedmitogenic factor (HIMF/FIZZ1/Relma) antibodies (Abcam),anti-interleukin-10 (Abcam) and anti-interleukin-6 (IL-6) antibodies(Santa Cruz) for overnight at 4° C. To detect mouse immunoglobulin A(IgA) as a loading control, 1:5,000 diluted goat anti-mouse IgA antibody(Abcam) was used. Peroxidase-conjugated anti-rabbit secondary antibody(Santa Cruz) was used in 1:50,000 dilution to visualize immunoreactivebands either by the enhanced chemiluminescence reagent (Pierce) orLumi-Light^(PLUS) (Roche).

Animals and hypoxic exposure. 8-week-old FVB male mice were eitherobtained from Charles River Laboratories (Wilmington, Mass.) or wereraised in the Animal Facility at Children's Hospital Boston. Mice ineach group were exposed to 8.5% oxygen in a Plexiglas chamber(OxyCycler, BioSpherix, Redfield, N.Y.) for variable experimentalperiods. Ventilation was adjusted to remove CO₂ so that it did notexceed 5,000 ppm (0.5%) (average range 1,000-3,000 ppm). Ammonia wasremoved by ventilation and activated charcoal filtration through an airpurifier. All animal protocols were approved by the Children's HospitalAnimal Care and Use Committee.

Hypoxia-induced acute lung inflammation mouse model. Mice were injectedthrough left jugular veins with either conditioned medium (40 μg/kg) orexosomes (4 μg/kg) or exosome-free conditioned medium (4 μg/kg). As thecontrol, 50 μl of PBS or culture medium were injected in parallel. 3hours after injection, mice were continuously exposed to monobarichypoxia (8.5% O₂) for the noted experimental periods. In the time-courseexperiment, additional injection of MEX was performed on the rightjugular veins at 4 days after hypoxic exposure.

Hypoxia-induced PAH mouse model. Mice injected with exosomes or controlsat day 0 and at 4 days after hypoxic exposure were continuously exposedto hypoxia for entire 3 weeks then anesthetized with pentobarbital (50mg/kg, i.p.). Right ventricular systolic pressure (RVSP) was measuredusing a closed chest approach and the PowerLab system (ADlnstruments,Colorado Springs, Col.), as previously described². After pressuremeasurements, lungs were perfused with PBS and inflated with 4%paraformaldehyde to fix the lung architecture. The fixed lungs were thenparaffin embedded and sectioned for immunohistochemical analysis. Heartswere immediately analyzed for Fulton's Index measurements (ratio betweenright ventricular weight and left ventricle plus septum weight,RV/[LV+S]), an assessment of right ventricular hypertrophy.

Bronchoalveolar lavage and counting alveolar macrophages. Animals wereanesthetized with 2,2,2-Tribromoethanol (250 mg/Kg i.p.) and theirtrachea were cannulated and blunt ended needle was installed.Bronchoalveolar lavage fluid (BALF) was collected via sequentialadministration of PBS (0.8 ml, 0.8 ml, 0.8 ml, and 0.9 ml) andapproximately 3 ml of individual BALF was recovered. Cells in BALFs werecollected by centrifuge at 400×g for 5 minutes and resuspended in Kimurastaining solution to selectively count total alveolar macrophages inBALFs.

Immunohistochemical analysis. Lung tissue sections were deparaffinizedin xylene and rehydrated on slices. Immunohistochemical analysis wasperformed by incubating with monoclonal anti mouse α-SMA antibody(Sigma) at a dilution of 1:125 overnight at 4° C. after block thetissues for 1 hour. After inactivating endogenous peroxidase with 3%H₂O₂ in methanol (Sigma), secondary antibodies, and peroxidase stainingwas performed according to manufacturer's instructions (Vectorlaboratories, Burlingame, Calif.). Vessel wall thickness was assessed bymeasuring α-SMA staining in vessels less than 30 μm in diameter withinsections captured under 400× magnification.

Isolation of human MSCs from human umbilical cord Wharton's Jelly. Humanumbilical cord Wharton's jelly derived MSCs (hUC-MSCs) were isolatedaccording to published methods (Mitchell, K. E. et al., 2003, Stem Cells21:50-60; and Penolazzi, L. et al., 2011, J Cell Physiol) with minormodifications. Cord was rinsed twice with cold sterile PBS, cutlongitudinally, and arteries and vein were removed. The soft gel tissueswere scraped out, finely chopped (2-3 mm²) and directly placed on 100 mmdishes (15 pieces per dish) with DMEM/F12 (1:1) (Invitrogen)supplemented with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine,and penicillin/streptomycin, and incubated for 5 days at 37° C. in ahumidified atmosphere of 5% CO₂. After removal of tissue and medium, theplates were washed 3 times with PBS, the attached cells were culturedand fresh media replaced 3 times per week. At 70-80% confluence, cellswere collected and stained with PE conjugated antibodies for CD34(Miltenybiotec) and CD45 (Miltenybiotec). Immunodepletion was performedusing the anti-PE-microbeads (Miltenybiotec) and MSCS column(Miltenybiotec) according to manufacturer's instructions. The CD34 andCD45 negative populations were further propagated and selected for theexpression of MSC markers (CD105, CD90, CD44, and CD73) and the absenceof CD11b, CD19, and HLA-DR by using a set of fluorescently-labeledantibodies specific for the characterization of human MSCs (BDBiosciences) using a MoFlo flow cytometry (Beckman Coulter).

Preparation of conditioned media. To exclude contamination fromserum-derived microvesicles, serum used for propagation of cell culturesand the collection of conditioned media was clarified byultracentrifugation at 100,000×g for 18 hrs. MSC were cultured in α-MEMmedia supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone) and10% (v/v) Horse Serum (Hyclone). MLFs were cultured in Dulbecco minimalessential medium (DMEM, Invitrogen) supplemented with 10% FBS and 2 mML-glutamine (GIBCO). Cultures at 70% confluence were washed twice withPBS and incubated with serum-free media supplemented with 2 mML-glutamine for 24 hours under standard culture conditions. Conditionedmedia were collected and cells and debris were removed by differentialcentrifugations at 400×g for 5 min, at 2,000×g for 10 min, and at13,000×g for 30 min. The clarified conditioned media were subsequentlyfiltered through a 0.2 μm filter unit and concentrated using aUltracel-100K (Millipore) centrifugal filter device, to a proteinconcentration range of 0.1-0.5 mg/ml. Protein levels in the conditionedmedia were determined by Bradford assay (Bio-Rad).

In vitro hypoxia. Human PAECs were purchased from GIBCO and cultured inM200 medium supplemented with LSGS (Invitrogen). At 80% confluence,cells were exposed to 1% O₂ for 5 hours in an inVivo2 workstation(Ruskin Technology, Bridgend, UK) in the presence or absence of exosomalfraction (1 μg/ml), or the exosome-depleted fraction of hUC-MSCconditioned media (1 μg/ml). Cells were lysed and proteins in whole celllysates were separated on 8% SDS-polyacrylamide gel electrophoresisfollowed by western blot analysis for phospho-STAT3 and STATS (CellSignaling).

Isolation of exosomes. 50 μl of concentrated conditioned media wasapplied on a CHROMA SPIN S-1000 column (Clontech) pre-equilibrated witha buffer containing 20 mM sodium phosphate (pH 7.4) and 300 mM NaCl.Each fraction (0.1 ml) was sequentially collected by gravity. For alarge scale preparation, 1.5 ml of clarified and concentratedconditioned media was injected on a column of 16/60 Hiprep SephacrylS-400 HR pre-equilibrated in the above buffer using an AKTA purifierchromatographic system (GE Healthcare, Piscataway, N.J.). Fractions (1ml) were collected at a flow rate of 0.5 ml/min. Polystyrene nanospheresof 50 nm diameter (Phosphorex, Fall River, Mass.) were used as a sizereference and elution fractions corresponding to this standard'sretention volume were pooled and further analyzed.

Protein extraction and immunoblotting. BALF (3 ml) was centrifuged at420×g for 10 min and cell-free BALF supernatants were used for proteinanalysis. Equal volumes of BALF specimens from individual animals in thesame group were pooled (1 ml) and proteins precipitated overnight by 20%trichloroacetic acid (Sigma). A fraction equivalent to 30% of eachprotein pellet was dissolved n 1× sodium lauryl sulfate (SDS)-loadingbuffer was separated on a denaturing 15% polyacrylamide gel. Aftertransfer to 0.2 μm PVDF membranes (Millipore), blots were blocked with5% skim milk and incubated with 1:1,000 diluted rabbit polyclonalanti-monocyte chemoattractant protein-1 (MCP-1) antibody (Abcam),anti-hypoxia-induced mitogenic factor (HIMF/FIZZ1/Relma) antibody(Abcam) for overnight at 4° C. To detect mouse Immunoglobulin A, 1:5,000diluted goat anti-mouse IgA antibody (Abcam) was used.Peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz) wasused in 1:20,000 dilution to visualize immunoreactive bands either bythe enhanced chemiluminescence reagent (Pierce) or Lumi-Light^(PLUS)(Roche).

For analysis of proteins from whole lung tissue, frozen lung tissueswere chopped for 5 seconds by Polytron in cold PBS containing 2 mMPhenylmethanesulfonyl fluoride (Sigma) and centrifuged at 3,000×g for 3min. Chopped tissue pellets were washed twice with cold

PBS containing 2 mM PMSF by centrifugation at 3,000×g for 3 min eachtime and the white cleaned tissue pieces were subjected on the lysiswith RIPA buffer containing protease inhibitor cocktail (Roche) andphosphatase inhibitor cocktail (Thermo). 40 μg of lung tissue extractswere separated on 10-20% gradient gel (Invitrogen). Antibodies used inimmunoblotting were against MCP-1, HIMF, IL-6, vascular endothelialgrowth factor (Abcam), total STAT3, and phospho-STAT3 (Y705) (CellSignaling). For loading control, mouse monoclonal (3-actin antibody(Sigma) was used. Exosome preparations were separated on 12%polyacrylamide gel and then transferred onto 0.45 μm PVDF membrane(Millipore). Goat polyclonal anti-CD63 (1:1,000; Santa Cruz) antibody,polyclonal rabbit anti-CD81 (1:1,000, Santa Cruz), and monoclonalanti-Dicer (1:1,000, Abcam) were used. To visualize the specific proteinbands, same ECL reagents described above were used. The ImageJ programfrom NIH was used for quantitation through densitometric analysis afterappropriate background subtraction.

Quantification of microRNAs. Total lung RNA was extracted by the methodof Chomczynski & Sacchi (1987 Anal Biochem 162:156-159) and 750 ng wasused as a template for reverse transcriptase with specific primers foreach target microRNA (TaqMan Reverse Transcription Kit, AppliedBiosystems, Foster City, Calif.). Each reverse transcription reactionincluded also the primer for the small nuclear RNA sno202, which wasused as an internal control. 37.5 ng cDNA was used for each 20 μl qPCRreaction with TaqMan universal master mix II with no UNG (AppliedBiosystems) in the presence of probes specific for the indicatedmicroRNAs and the internal control (TaqMan microRNA assay, AppliedBiosystems). Amplification was performed at 50° C. for 2 min, 95° C. for10 min, followed by 40 cycles of 95° C. for 15 sec, 60° C. for 1 min, ona StepOne Plus platform (Applied Biosystems).

Results

BM-MSC secrete factors that suppress hypoxia-induced acute inflammatoryresponses. Therapeutic capacity of BM-MSCs have been observed fromseveral animal models of lung injuries. We first determined BM-MSCs wererelevant to hypoxia-induced pulmonary inflammation by their paracrinemanner. Hypoxic exposure results in significant pulmonary accumulationof macrophages and elevation of proinflammatory mediators within 2days². To test paracrine potentials of BM-MSCs on this animal model,mice receiving either BM-MSC-conditioned medium (BM-MSC-CM) or vehicleor MLF-conditioned medium (MLF-CM) were exposed to monobaric hypoxia for2 days. Consequently, hypoxia-derived acute pulmonary influx ofmacrophages was blocked by BM-MSC-CM treatment while mice injected withvehicle or MLF-CM showed a significant accumulation of macrophages inlung (FIG. 1A), suggesting BM-MSCs secrete factor(s) suppresshypoxia-derived lung inflammatory responses which signal to recruitmacrophages into the lung. As it has been observed that hypoxicconditioning upregulates pulmonary levels of proinflammatory mediators,cell-free BALFs from the mice were applied to comparative analysis forhypoxia-responsible proinflammatory mediators, MCP-1 and HIMF/FIZZ1. Invehicle or MLF-CM injected mice, secretion levels of both MCP-1 and HIMFin the lung were significantly increased by hypoxic exposure for 48hours. In contrast, the elevation of these mediators by hypoxia waseffectively suppressed in BM-MSC-CM treated mice (FIG. 1B). Takentogether, secretory factor(s) of BM-MSCs are anti-inflammatory agentswhich prevent pulmonary recruitment of macrophages via blocking thehypoxia-induced upregulation of MCP-1 and HIMF/FIZZ1 in the lung.

BM-MSC secrete exosomes. We isolated small vesicles in BM-MSC-CM by aprocedure including ultrafiltration and size-exclusion chromatography.Table 2 shows the degree of enrichment achieved in these experiments. Assummarized in FIG. 2, approximately 1.6% (w/w) of secretory proteins inBM-MSC-CM might be associated with their exosomes. MLF-derived exosomes(FEX) were isolated as a control and analyzed in parallel. From theelectron microscopic analysis, exosomes were observed only in thefraction within void volume of the column, suggesting that sizeexclusion chromatography to exclude molecules smaller than 8,000 kDa ishighly selective to enrich exosomes (FIGS. 2A, 2B). Moreover, electronmicrographs of exosomes from the equivalent fraction of BM-MSC-CM andMLF-CM confirmed that exosomes shed from the both types of cellsdemonstrated physical parameters of typical exosomes such asheterogeneity in diameter ranging from 30 to 100 nm and biconcavemorphological characteristics (FIGS. 2C, 2D). With regard to proteincontent of BM-MSC-derived exosomes (MEX), western blot analyses showedthat MEX were positive for typical exosomal proteins such as CD63 andmoesin, and also highly associated with immunomodulatory proteinsincluding monocyte colony stimulating factor (mCSF) and osteopontin(OPN/SPP1). Some isoforms of 14-3-3 family, which are small polypeptideswith a molecular mass of approximately 30 kDa capable of bindingnumerous functionally diverse signaling proteins, co-purified withexosomes indicating that a certain subset of 14-3-3 isoforms isassociated with MEX. Moreover, Dicer which catalyzes a criticalprocessing step of microRNA maturation in cytoplasm was only detected inthe exosomal fraction, strongly supporting that microRNAs are anotherconstituent of exosomes. It is interesting to note that mCSF and OPN aswell as CD63 and moesin were also abundantly detected in exosome-freefractions obtained during the purification procedure, suggesting thepresence of their soluble isoforms in the exosome-free fraction or weakor low affinity association with the surface of exosomes (FIG. 2E).Comparative western analysis revealed that MEX are highly enriched inCD63, Dicer, mCSF, and osteopontin as compared to FEX while CD81 is moreabundant in FEX (FIG. 2F). Consequently, MEX preserve physicalcharacteristics of typical exosomes in terms of size and morphology andwere highly enriched with Dicer and immune modulators compared with FEX.We further performed comparative proteomic analyses between the exosomesfrom the two different cell types by mass spectrometry to furtherinvestigate the physiological roles of MEX.

Anti-inflammatory roles of BM-MSCs were mediated by their secretoryexosomes. We further investigated whether BM-MSC-derived exosomes arephysiologically functional in the experimental model of hypoxia-inducedacute pulmonary inflammation. Mice injected with purified MEX wereexposed to monobaric hypoxia of 8.5% O₂. After continuous exposure tohypoxia for 48 hours, we observed that hypoxia-derived pulmonary influxof macrophages was effectively prevented by administration of MEX. Incontrast, FEX or exosome-free fraction of BM-MSC-CM failed to preventthe pulmonary influx of macrophages (FIG. 3A). Total proteins fromcell-free BALFs were studied using immunoblot analysis. Upregulation ofsecretory proinflammatory mediators such as MCP-1 and HIMF/FIZZ-1 byhypoxia were completely abrogated by administration of MEX, while thesewere not blocked by injection of vehicle or FEX (FIG. 3B).Interestingly, exosome-free fraction of BM-MSC-CM failed to suppresshypoxia-induced upregulation of these proinflammatory mediators. Therewere few other differences in protein contents between the exosomalfraction and exosome free fraction, suggesting the possibility thatnucleic acids in exosomes may be important in the response. These datahighlight that BM-MSC-derived secretory factors specifically localizedon exosomes effectively suppress hypoxia-induced pulmonary inflammatoryresponses by blocking the hypoxia-derived signal to upregulateproinflammatory mediators MCP-1 and HIMF/FIZZ 1.

Administration of MEX abrogates hypoxia-induced lung inflammatoryresponses. We observed that BM-MSC secrete exosomes which abrogatehypoxic signals to recruit macrophages into the lung, and also observedthat hypoxic exposure leads to acute inflammatory responses in the lungwithin 2 days. We further investigated the time course of single ormultiple treatments of MEX on pulmonary inflammatory responses until 7to 11 days of hypoxic exposure. In vehicle injected group, miceexhibited acute pulmonary influx of macrophages and dramatic elevationof pulmonary level of both MCP-1 and HIMF/FIZZ1 by 2 days of hypoxicexposure with the inflammatory peak resolving at 7 days of hypoxicexposure. Unlike with reductive number of alveolar macrophages andpulmonary level of MCP-1, high level of HIMF/FIZZ1 was sustained for 7days of continuous hypoxic exposure, suggesting MCP-1 is mainlyregulating pulmonary influx of macrophages while HIMF/FIZZ1 might playdistinct roles in the response to hypoxia (FIGS. 4A, 4D). Importantly, asingle injection of

MEX was not able to suppress hypoxia-induced inflammatory responses morethan 4 days under hypoxia, so hypoxia-responsible pulmonary inflammationwas initiated after 4 days of injection and peaked at 7 days thenresolved at 11 days (FIGS. 4B, 4D). More importantly, additionalinjection of MEX at the 4^(th) day of hypoxic exposure sustained theblockade of pulmonary inflammation under hypoxia up to 11 days (FIG.4C). With regard to HIMF/FIZZ1 regulation by MEX, a single injection ofMEX is able to suppress hypoxia-induced upregulation of HIMF/FIZZ1 for 4days of hypoxia. Additional injections of MEX were not able to abrogateupregulation of HIMF/FIZZ1 at 7 days of hypoxia, suggesting othertemporal regulatory pathway might be involved in this response. Takentogether, hypoxia-induced acute pulmonary inflammation was temporallysuppressed by a single injection of MEX and the anti-inflammatoryeffects able to neutralize pulmonary response to hypoxia were maintainedby sequential and multiple administration.

Hypoxia-induced PAH suppressed by BM-MSC-derived exosomes. In thisstudy, we observed that MCP-1 and HIMF were significantly upregulated byhypoxia in the lung and that the hypoxia-induced upregulation wasmarkedly attenuated by treatment of MEX. Therefore, we hypothesized thatMEX might prevent hypoxia-induced PAH by blocking both importantmediators of PAH. To test the hypothesis, mice were exposed to hypoxiafor 3 weeks after receiving either MEX or FEX or PBS as control. At theend of experimental period, RVSP was measured and heart tissue wasprocessed for RV hypertrophy. FIGS. 5B and 5C showed that all thehypoxic mice exhibited elevated RVSP and Fulton's Index compared withage-matched normoxic mice. In contrast, significant improvement wasobserved for the mice that received MEX as compared to the mice thatreceived either PBS or FEX. Moreover, compared with mice that received asingle injection of MEX, mice that received additional injections of MEXat day 4 showed significantly reduced RVSP and RV hypertrophy underchronic hypoxia, indicating repeated administration of MEX amelioratespulmonary artery pressure and ventricular wall thickness in response tochronic hypoxia. To investigate whether multiple treatments of MEX couldattenuate hypoxia-induced pulmonary vascular remodeling, histologicalsections of the hypoxic lungs were morphometrically analyzed by stainingpulmonary vessels with alpha-SMA antibody (FIG. 5D). The percentage ofmedial vessel wall thickness of small pulmonary arterioles within arange of 20˜30 μm in diameter was determined. In comparison withage-matched normoxic control mice, markedly increased thickness of smallpulmonary arterioles by chronic hypoxia was observed in either PBS orFEX treated mice while no significant difference was observed for thevessel wall thickness between the control and MEX treated mice,indicating that MEX are able to prevent the process of hypoxia-inducedpulmonary vascular remodeling (FIG. 5E).

MEX comprise a variety of immunomodulatory factors. We have observeddramatic effects of MEX on both hypoxia-induced acute pulmonaryinflammation and pulmonary artery hypertension by chronic hypoxia. Toinvestigate their molecular mechanism, we performed global proteomicprofiling of both MEX and FEX by high performance liquid chromatographymass spectrometry (HPLC-MS/MS). A total of 273 proteins were identifiedwith high confidence in MEX and 35% of proteins were also detected inFEX. To achieve high confidence for profiling considerable proteinsassociated with MEX, we identified proteins with high (>25) number ofMS/MS spectra and high (>3) ratio of MEX/FEX in sequence coverage. 8proteins fit this criterion and these are listed in Table 1. Among theseproteins, 3 were unique and 5 were highly enriched in MEX.Galectin-3-binding protein (LGALS3BP/MAC2BP), which is one of the uniqueproteins in MEX, is a secretory protein that has been shown to possessimmunomodulatory activities by inhibiting transcription of TH2 cytokinewhich is hallmark of athma³⁴. It is able to interact with a variety ofproteins on the cellular surface and matrix including the lectin family,integrins, laminins, and fibronectin. As the interactions have beenimplicated in modulating tumor cell adhesion to extracellularproteins³⁵, GAL3BP on the surface of MEX might play an important role totarget the infused MEX to the surface of recipient cells in a ligandspecific manner. Another unique protein in MEX, thrombospondin-2, isknown to act as a potent endogenous inhibitor of tumor growth andangiogenesis³⁶ and to suppress the production of pro-inflammatorycytokines IFN-γ and TNF-α³⁷. Lactadherin (MFGE8), a major component ofdendritic cell-derived exosomes³⁸, has been reported to play a role incell death and apoptosis where it recognizes specificallyphosphatidylserine exposed on apoptotic cells and promotes phagocyticclearance of apoptotic cells by binding to cells expressingintegrin_(α)v and integrinp₆₃ ^(39,40). On the surface of MEX,lactadherin may be involved in targeting MEX to their recipient celltypes. Moreover, it has been reported that lactadherin is also involvedin phagocytic clearance of amyloid beta-peptide (Abeta), which is amajor component in accumulated senile plaques in Alzheimer's disease, bydirect protein-protein interaction. The abundance of Abeta in exosomalfraction is possibly due to direct interaction between lactadherin andAbeta. Adipocyte-enhancer-binding protein 1(AEBP1), also called aorticcarboxypeptidase-like protein (ACLP), plays important physiologicalroles in wound healing and energy homeostasis. Mice lacking exons 7-16exhibit deficient wound healing and AEBP1-null mice are resistant todiet-induced obesity⁴¹. Table 1 and FIG. 22 describe the variousmediators identified in mouse and human MEX.

MEX of either mouse or human origin mediate the suppression of STAT3activation by hypoxia. Early hypoxia resulted in activation of STAT3 inthe mouse lung, through phosphorylation at Tyr-705, and without anyeffect on the total levels of STAT3 protein. This activation wasefficiently suppressed by MEX treatment (FIG. 19A). STAT3 is atranscription factor integral to signaling pathways of many cytokinesand growth factors and STAT3 activation plays a critical role inrespiratory epithelial inflammatory responses. Importantly, persistentex vivo STAT3 activation, has been linked to the hyperproliferative andapoptosis-resistant phenotype observed in PAECs (Masri, F. A. et al.,2007, Am J Physiol Lung Cell Mol Physiol 293:L548-554) and pulmonaryartery smooth muscle cells (PASMCs) (Paulin, R. et al., 2011,Circulation 123:1205-1215) from patients with idiopathic pulmonaryarterial hypertension (IPAH). Therefore, suppression of hypoxic STAT3activation could account for the pleiotropic protective effects of MEXtreatment.

To verify that the suppression of this hypoxic signaling is not aproperty specific to MEX of mouse origin, MSCs from human umbilical cordstroma (hUC-MSC) (Mitchell, K. E. et al. and Penolazzi, L. et al.) wereisolated and exosome-enriched (hUC-MEX) and exosome-depleted(hUC-ExD-CM) fractions were prepared from hUC-MSC conditioned mediathrough size exclusion chromatography, as described herein. As depictedin FIG. 19B, exposure of hPAECs to hypoxia results in robust activationof STAT3 by Tyr-705 phosphorylation. Treatment with hUC-MEX completelyabrogated this response, whereas the fraction depleted of microvesicleshad no effect. In addition to demonstrating that suppression of STAT3activation is a property shared by MEX of both human and mouse origin,these results strongly suggest that direct suppression of hypoxicsignaling in pulmonary vascular cells is a primary function underlyingthe protection conferred by MEX treatment.

MEX treatment suppresses the hypoxic induction of the miR-17 microRNAsuperfamily and increases levels of anti-proliferative miR-204 in thelung. STAT3 (activated by either VEGF or IL-6) has been reported todirectly regulate the transcription of the miR-17-92 cluster ofmicroRNAs in PAECs, resulting in decreased levels of bone morphogeneticprotein receptor-2 (BMPR2), a target of miR-17 (Brock, M. et al., 2009,Circ Res 104:1184-1191). Therefore, we assessed the effect of hypoxiaand MEX treatment on the miR-17-92 cluster of microRNAs and itsconserved paralog clusters, miR-106b-25 and miR-106a˜363. These microRNAclusters have been postulated to be pro-proliferative, targeting anarray of genes involved in the G1/S phase transition (Cloonan, N. etal., 2008, Genome Biol 9:R127) and have been reported to play a centralrole in embryonic lung morphogenesis (Carraro, G., 2009, Dev Biol333:238-250). We found that select microRNAs representing all threeclusters of the miR-17 superfamily were upregulated by hypoxia in thelung, and this transcriptional activation was efficiently suppressed byMEX treatment (FIG. 20A). Interestingly, levels of microRNAs involved inhypoxic signaling networks, such as miR-199a-5p, a microRNA reported tostabilize HIF1α in cardiac myocytes (Rane, S. et al., 2009, Circ Res104:879-886), miR-214, which shares the same host gene with miR-199(Watanabe, T. et al., 2008, Dev Dyn 237:3738-3748), or miR-210, ahypoxamir under direct HIFla regulation (Chan, S. Y. et al., 2010, CellCycle 9:1072-1083), were not affected by MEX treatment (FIG. 20B),pointing to targeted effects of MEX on specific hypoxia-regulatedsignaling pathways.

Importantly, we observed that MEX treatment resulted in the increase oflung levels of miR-204, (FIG. 20C) a microRNA enriched in distalpulmonary arteries that is transcriptionally suppressed by STAT3 butalso inhibits the activation of STAT3 in a feed-forward regulatory loop(Courboulin, A. et al., 2011, J Exp Med 208:535-548). The proliferativeand anti-apoptotic phenotype of PASMCs isolated from patients with IPAHis inversely related to the level of miR-204 and delivery of exogenousmiR-204 to the lungs of animals with PH ameliorated established disease.Therefore, we interpret these results as an indication that MEXtreatment, by suppressing STAT3 activation at the early stages ofhypoxic exposure, prevents the hypoxic induction of thepro-proliferative miR-17 superfamily in the lung vasculature and blocksthe STAT3-miR-204-STAT3 feed-forward loop in distal pulmonary vessels.This shifts the balance towards an anti-proliferative state in the lungvasculature and prevents vascular remodeling under chronic hypoxia. FIG.21 is a schematic representation of the hypoxic signaling pathwaysproposed to be operative in the development of PH that are modulated byMEX.

In summary, MSC-conditioned media was fractionated throughsize-exclusion chromatography to identify the biologically-activecomponent protecting against hypoxia-induced lung inflammation and HPH.It was found that MEX are the critical vectors of MSC action: MEXefficiently suppressed the hypoxic pulmonary influx of macrophages andblocked the upregulation of the pro-inflammatory and mitogenic mediatorssuch as MCP-1, IL-6, and hypoxia-induced mitogenic factor (HIMF;FIZZ1/RELM-α/RETNLA) in the hypoxic lung. Pro-proliferative pathwaysactivated in the hypoxic lung were also blocked by MEX treatment, asevidenced by the suppression of signal transducers and activators oftranscription (STAT3).

This resulted in increased lung levels of miR-204, a microRNA enrichedin distal pulmonary arterioles that is down-regulated in both human PHand in experimental models of disease (Courboulin, A. et al.). It wasalso found that hypoxia upregulates members of the miR-17 family ofmicroRNA clusters in lung tissue, microRNAs shown to be under theregulatory control of STAT3, and that MEX treatment efficientlysuppresses this pro-proliferative signal. MEX isolated from the culturemedia of human umbilical cord-derived MSCs had similar inhibitory effecton hypoxic proliferative signaling pathways as the mouse MEX. Human MEXsignificantly inhibited the hypoxic activation of STAT3 in culturedhPAECs. In contrast, exosome-depleted MSC-culture media had nophysiologic effect in vivo nor on cultured cells in vitro, pointing toMEX as the key effectors of MSC paracrine function.

TABLE 2 Purification of MSCs-derived exosomes Total Step VolumeConcentration protein (mg) Yield (%) Serum-free 25 28.91 7,228 100MSCs-conditioned Ultrafiltration 1 7,184.70 7,185 99.4 (100 kDa MWCO)S-400 column 4.5 166 747 10.4 chromatography

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EQUIVALENTS

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

1-8. (canceled)
 9. A method comprising administering to a subject havingor at risk of developing a lung disease an effective amount of isolatedmesenchymal stem cell (MSC) exosomes. 10-15. (canceled)
 16. The methodof claim 9, wherein lung disease is inflammatory lung disease, lungvascular disease, or acute lung injury.
 17. The method of claim 16,wherein the inflammatory lung disease is pulmonary hypertension, asthma,bronchopulmonary dysplasia (BPD), allergy, or idiopathic pulmonaryfibrosis.
 18. The method of claim 16, wherein the acute lung injury isassociated with sepsis or is ventilator-induced acute respiratorydistress syndrome (ARDS).
 19. The method of claim 9, wherein the subjecthas or is likely to develop schistosomiasis.
 20. The method of claim 9,wherein the subject is an neonate.
 21. The method of claim 9, whereinthe subject is an infant. 22-23. (canceled)
 24. The method of claim 9,wherein the subject was born prematurely. 25-27. (canceled)
 28. Themethod of claim 9, wherein the isolated MSC exosomes are used togetherwith a secondary agent.
 29. The method of claim 28, wherein thesecondary agent is a steroid, an antioxidant, or inhaled nitric oxide.30-33. (canceled)
 34. The method of claim 20, wherein the isolated MSCexosomes are administered within 1 month of birth.
 35. The method ofclaim 9, wherein the isolated MSC exosomes are administeredintravenously.
 36. The method of claim 9, wherein the isolated MSCexosomes are administered to lungs or trachea of the subject.
 37. Themethod, composition, use, or isolated MSC exosomes of claim 36, whereinthe isolated MSC exosomes are administered by inhalation. 38-44.(canceled)
 45. The method of claim 9, wherein the isolated MSC exosomesare administered repeatedly to the subject.
 46. (canceled)
 47. Themethod of claim 9, wherein the isolated MSC exosomes are administeredcontinuously to the subject.
 48. The method of claim 9, wherein theisolated MSC exosomes are derived from cord blood MSC.
 49. The methodclaim 9, wherein the isolated MSC exosomes are derived from bone marrowMSC.
 50. (canceled)
 51. The method of claim 9, wherein the isolated MSCexosomes are allogeneic to the subject.
 52. The method of claim 9,wherein the subject is not receiving a cell or organ transplantation.53-58. (canceled)